Protective Eflects Of Astra Galoside IV On High Insulin Induced Human Imesangial Cells Injury And Its Mechanisms

Objective To investigate the effects of insulin at different concentrations on human mesangial cells( HMCs) proliferation and to research the protective effects of Astragalosides IV( As-IV) on high insulin induced HMCs injury in vitro. To discuss the possible mechanism on the development and progression of diabetic nephropathy( DN) and to explore the molecular mechanisms of As-IV in preventing HMCs injury, in order to offer theoretical basis for DN prevention and As-IV clinical application.Methods 1. The HMCs were cultured with DMEM medium containing 10% fetal calf serum, and incubated at 37 ℃with 5% CO2 in the constant temperature incubator. The HMCs at logarithmic growth phase were digested with 0.25% trypsin and taken for the subsequent experiment. 2. The cells were randomly divided and treated as follows:(1) normal group(NG), insulin(INS, respectively treated with 1, 2, 4, 8, 16, 32, 64, 128 nmol/L insulin) group;(2) normal group(NG), high insulin group(HINS, 64 nmol/L), DPI(10 μmol/L), Tempol(100 μmol/L), LY294002(10 μmol/L) and As-IV(6.25, 12.5, 25, 50, 100 μmol/L);(3) normal group(NG), high insulin group(HINS, 64 nmol/L), Tempol(100μmol/L), LY294002(10 μmol/L) and As-IV(25, 50, 100 μmol/L);(4) normal group(NG), high insulin group(HINS, 64 nmol/L), DPI(10 μmol/L), Tempol(100 μmol/L), LY294002(10 μmol/L) and As-IV(25, 50, 100 μmol/L);(5) normal group(NG), high insulin group(HINS, 64 nmol/L), DPI(10 μmol/L), LY294002(10 μmol/L) and As-IV(25, 50, 100 μmol/L). 3. The cells were randomly divided and treated as 2(1), MTT assay was applied to detect the value of OD at different concentrations and time. According to the results, the optimal concentration and time of insulin were chosen for the following experiment. Then the cells were randomly divided and treated as 2(2), MTT assay was also applied to detect the value of OD. Optimal drug concentration is selected for the subsequent experiment at the same time. 4. The cells were randomly divided and treated as 2(3), then making use of enzyme-linked immunosorbent assay(ELISA) to test the contents of fibronectin(FN), tissue inhibitor of metalloproteinase-1(TIMP-1) and matrix metalloproteinase-9(MMP-9). At the same time, the cells were randomly divided and treated as 2(4), and using the 2,7-dichlorodihydro fluorescin diacetate(DCFH-DA) to detecte the amounts of reactive oxygen species(ROS). Furthermore, the expression of NADPH oxidase 4(NOX4) in 2(5), phosphor-protein kinase b(p-PKB, also named p-Akt) and phosphor-mammalian target of rapamycin(p-m TOR) in 2(3) are analyzed by using Western blot.Results 1. Acoording to the MTT assay results, increased the concentrations and time of insulin, both can increase the proliferation of HMCs, and the effect was in a time-andconcentration-dependent manners. HINS could increase HMCs proliferation after a certain period of 48 hours contrasted with NG group(P<0.01). Compared with HINS group, As-IV(6.25, 12.5, 25, 50, 100 μmol/L) and DPI, Tempol, LY294002 could inhibit the proliferation of HMCs in varying degrees(P<0.05, P<0.01). As-IV inhibited HMCs proliferation was in a concentration-dependent manner, and As-IV(25, 50, 100 μmol/L) inhibitory effect was statistically significant(P<0.05, P<0.01). 2. ELISA results show that compared with NG, the contents of FN, MMP-9, TIMP-1 in HMCs induced by high insulin increased significantly, but the ratio of MMP-9/TIMP-1 was low(P<0.01). Compared with HINS group, Tempol, LY294002, and As-IV(25,50,100 μmol/L) could improve the inappropriate secretions of FN, MMP-9, TIMP-1 induced by high insulin(P<0.05, P<0.01), and As-IV show a concentration-dependent manner. The results show that As-IV could regulate the synthesis and degradation of extracellular matrix components in HMCs. 3. The mean DCF fluorescence intensity results show that there were some expressions of ROS in NG. But compared with the NG, HMCs cultured in high insulin medium enhanced obviously(P<0.01), which suggested that the levels of intracellular ROS in HINS group increased significantly. DPI, Tempol, LY294002 and As-IV( 25,50,100 μmol/L) could weaken the fluorescence intensity in varying degrees(P<0.05, P<0.01), which show that all treatment groups could reduce the intracellular ROS contents and remit the oxidative stress by high insulin. 4. Form the results of Western blot, compared with the NG, the contents of NOX4、p-Akt、p-m TOR proteins increased in HINS group(P<0.01). DPI, LY294002 and As-IV(25,50,100 μmol/L) treatments decreased the expressions of NOX4; and also Tempol, LY294002 and As-IV(25,50,100 μmol/L) treatments decreased the expressions ofp-Akt, p-m TOR proteins cultured in high insulin medium(P<0.05, P<0.01). The effect of As-IV(25,50,100 μmol/L) was in a concentration-dependent manner.Conclusion Insulin can promote the proliferation of HMCs cultured in vito, and the effect was in a time-and concentration-dependent manners. As-IV has protective effects on HMCs injured by high insulin, and the effect was in a concentration-dependent manner. The protective mechanism of As-IV may on the basis of inhibiting the cell proliferation, decreasing the high expression of NOX4, p-Akt, p-m TOR proteins, regulating the inappropriate secretion about FN、MMP-9、TIMP-1 and reducing the state of cellular oxidative stress.